1. Field of the Invention
The present invention relates to a process for culturing cells by introducing the cells into the bone or bone marrow of a mammal. The invention also relates to a method for delivering a biologically active substance to a mammal by introducing the substance into the bone or bone marrow.
2. Discussion of the Background
The bone marrow (BM) is an organ present between the cortical walls of bone. In infancy and childhood, the bone marrow of the entire body is involved in hematopoiesis. In the adult, hematopoiesis is limited to axial skeleton (skull, sternum/ribs, vertebral bodies, iliac crests).
The BM provides access to the circulatory system, and additionally to hematopoietic stem cells and stromal cells. In the past, intra-osseous bone infusions were used as a method for pediatric resuscitation when an IV was not easily accessible. This approach was abandoned with the advent of improved venous access devices. Later, the technique underwent a revival in pediatric trauma and burn victims [(Evans et al, Burns 21(7):552-3 (1995); Hopkins et al, Jrnl of the Louisiana State Medical Society 142(3):31-2, 1990)].
That the bone marrow may be an immunoprivileged site is supported by the literature. Paramithiotis and Cooper showed that antigen-experienced B lymphocytes do not secrete Ig spontaneously when removed from the bone marrow, even though this occurs when these lymphocytes are removed from other locations. Yokichi et al disclosed the decreased responsiveness of bone marrow macrophages versus peripheral macrophages to LPS stimulation. This includes a decreased bone marrow macrophage response even in the face of pre-sensitization with LPS. Hirsch et al describes the immunosuppressive effects of TGFxcex2 in the T cell response to TB. Because TGFxcex2 is present in large amounts in the bone and thus the bone marrow. These reports are consistent with a theory that the bone marrow micro-environment is immunosuppressive.
Other studies have shown the utility of intra-osseous infusions for withdrawing laboratory samples [(Orlowski et al, Annals of Emergency Medicine 18(12):1348-51 (1989)] and for infusion of pharmacologically active compounds [(Cameron et al, Jrnl of Emergency Medicine 7(2):123-7 (1989); Neish et al, Am J of Diseases of Children 142(8):878-80 (1988); Brickman et al, Annals of Emergency Medicine, 16(10):1141-4 (1987)]. One such study measured the flow rates in a clavicular intra-osseous infusionxe2x80x94and found it to be statistically similar to sub-clavian vein flow, while the iliac crest was found to have a flow twice that of the sub-clavian vein [(Iwama et al, Jrnl of Medical Science 40(1):1-8 (1994)].
Sites in the body which are immunologically privileged can be utilized for the introduction of materials foreign to the host. Immune privilege may stem from either immune suppression, immune deviation, or active tolerance induction. Features characteristic of immune privileged sites include the presence of a blood tissue barrier, cytokines such as TGF-xcex2, neuropeptides, the presence of antigen presenting cells which actively induce tolerance, and other factors [(Streilin, Science 270(5239)1158-9 (1995)].
Immune privileged sites have developed in specific compartments of the body in which immune stimulation might be harmful to the host, due to the presence of tissue which may be seen as foreign. These sites are then compartmentalized in some way so as to create a micro-environment conducive to immune suppression and active immune tolerance.
An example of an immune privileged site is the pregnant uterus. The placenta and fetus represent foreign tissue to the mother, yet these antigens are under normal circumstances not rejected. The mother and fetus interconnect via sinusoids present between the placenta and uterus. During pregnancy, there are fetal blood cells present within the maternal circulatory system. These cells and fetus are not rejected so long as the compartmentalization of this foreign tissue and it""s interface with the non-immune privilege host, is not compromised. So long as the compartments remain whole, the microenvironment is conducive for immune suppression via local growth factors such as TGF-xcex2, presence of Fas-ligand, and ACAID (anterior chamber associated immune deviation).
Once the compartment is breached, as occurs during the separation of the placenta from the uterus, the status of immune privilege is breached, and the mother can mount an immune response against the fetus, which is the situation of an Rh negative mother with an Rh positive fetus. Throughout the initial pregnancy with and Rh positive fetus, there is no immune response to the Rh antigen. Once the compartment is breached following delivery, if Rhogam is not administered, the mother will develop a permanent response to Rh antigen, which may result in an attack on the next Rh positive fetus [(Tafuri et al, Science 270:630 (1995)].
As noted above, an immune privileged site may exist as a discrete xe2x80x9cmicroenvironment.xe2x80x9d The microenvironment in which an antigen exists or is present determines the host response to it. The phenotype of immunity is modulated by the range of environments that lymphocytes experience as they pass through, or lodge in for a time [Doherty, J. Immunol. 155(3):1023-7 (1995)].
To date, the use of bone marrow was mainly limited to the use of BM derived cells for use in hematopoietic transplantation. However, in 1968, Fonkalsrud described the possibility for using the bone marrow space as an immunologically-privileged site for allogeneic skin grafting (Surgery, Gynecology and Obstetrics, pp. 71-75). In 1969, Fonkalsrud again described the possibility of using the marrow space for implants, this time for thyroid allografts (Arch. Surg. Vol 98, pp. 738-741. However, there have been no reports of transplantation into the bone marrow of other types of cells, such as hepatocytes and pancreatic islet cells or for the use of the BM space for the delivery of peptides, drugs, genes nor for the use of the space for the culture of cells for use in transplantation such as hepatocytes, nervous, cardiac or other useful tissue. Ricordi (Pancreatic Islet Cell Transplantation, p. 317-319 (1992), which is incorporated herein by reference in its entirety) has shown that non-immunoisolated rat-to-mouse islet xenografts show poor survival, however, islets have not been transplanted into bone marrow.
In view of the aforementioned lack of utilization of the bone marrow for surgical intervention, there exists a need in the art for such a method.
Accordingly, one object of this invention is to provide a method for culturing cells comprising transplanting the cells into the bone marrow of a mammal.
Another object of the invention is to provide a method for transplanting cells into a mammal comprising delivering the cells into the bone marrow of a mammal.
Still another object of the invention is to provide a method for delivering a functional gene into a mammal comprising delivering a cell transformed with a vector containing the gene into the bone marrow of a mammal.
Yet another object of the invention is to provide a method for delivering a biologically active protein or peptide to a mammal, comprising delivering a cell transformed with a vector which expresses a gene encoding the protein or peptide into the bone marrow of a mammal.
Another object of the invention is to provide a method for delivering a biologically active protein or peptide to a mammal, comprising delivering a DNA encoding the protein or peptide to the bone marrow of a mammal.
A further object of the invention is to provide a method for delivering a biologically active protein or peptide to a mammal comprising delivering said protein or peptide in a suitable carrier to the bone marrow of a mammal.
A further object of the invention is to provide a method for delivering a pharmaceutical to a mammal comprising delivering the pharmaceutical in a suitable carrier to the bone marrow of a mammal.
Another object of the invention is to provide a method for performing bone marrow transplants.
Still another object of the invention is to provide a method for inducing tolerance to an antigen in a patient prior to treatment.
With the foregoing and other objects, advantages and features of the invention that will become hereinafter apparent, the nature of the invention may be more clearly understood by reference to the following detailed description of the preferred embodiments of the invention and to the appended claims.